Gradient peak capacity

The gradient peak capacity is significantly higher than the isocratic peak capacity, P , within the same time limits, as illnstrated in Figure 5.10. 

Peak capacity can be very effectively improved by using temperature programming in GC or gradient elution in LC. However, if the mixture is very complex with a large number of individual solutes, then the same problem will often arise even under programming conditions. These difficulties arise as a direct result of the limited peak capacity of the column. It follows that it would be useful to derive an equation that... 

The resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

The peak capacity is not pertinent as the separation was developed by a solvent program. The expected efficiency of the column when operated at the optimum velocity would be about 5,500 theoretical plates. This is not a particularly high efficiency and so the separation depended heavily on the phases selected and the gradient employed. The separation was achieved by a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. Initially the tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase. 

It is seen that a high peak capacity was available but, as a gradient program was used, the isocratic peak capacity is not pertinent. The mobile phase program started with a solvent mixture containing 20% v/v of ethyl acetate in n-hexane and ended with pure ethyl acetate. 

Dolan, J.W., Snyder, L.R., Djordjevic, N.M., Hill, D.W., Waeghe, T.J. (1999). Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations. J. Chromatogr. A 857, 1-20. 

Wang, X., Stoll, D.R., Carr, P.W., Schoenmakers, P.J. (2006). A graphical method for understanding the kinetics of peak capacity production in gradient hquid chromatography. J. Chromatogr. A 1125, 177-181. 

As mentioned earlier, high-speed separation is necessary to carry out fast, comprehensive 2D HPLC. The polymer monoliths have not been employed in such 2D HPLC, probably because permeability of polymer monoliths is not high enough to allow fast elution of the second dimension (2nd-D) in simple 2D operation, and the gradient cycle at the 2nd-D cannot be so fast to allow online 2D operation without reducing peak capacity at first dimension (lst-D). 

PEAK CAPACITY INCREASE BY USING MONOLITHIC SILICA COLUMNS IN GRADIENT ELUTION... 

A typical HPLC separation using a 15-cm column of 15,000 theoretical plates produces peak capacity (Giddings, 1991) of about 80-100 under isocratic conditions and up to 150 under gradient conditions in 1 h(Eq. 7.3, n peak capacity, A number of theoretical plates of a column, and fR and t retention time of the last and the first peak of the chromatogram, respectively). An increase in the number of separated peaks per unit time can be achieved by increased separation speed made possible by monolithic silica columns (Deng et al., 2002 Volmer et al., 2002). This has also been shown for peptides and proteins (Minakuchi et al., 1998 Leinweber et al., 2003). 

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